Folate Copolymer-Mediated Transfection of Cultured Cells

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Bioconjugate Chem. 1999, 10, 947−957

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Folate Copolymer-Mediated Transfection of Cultured Cells Christopher P. Leamon,*,† Debra Weigl,† and R. Wayne Hendren† Department of Oligomer Development, GlaxoWellcome Research Institute, 5 Moore Drive, Research Triangle Park, North Carolina 27709. Received May 27, 1999; Revised Manuscript Received August 3, 1999

Poly(ethylene glycol) of various sizes was used as a molecular spacer to separate the cell-targeting ligand, folate, from the surface of poly-L-lysine. The resulting ternary macromolecule (pLys-PEGfolate) was investigated in various formulations for its ability to transfect reporter plasmids into receptor-bearing HeLa and IGROV cell lines. Formulations were optimized with respect to DNA content, ( charge ratio, and the size and amount of PEG substitution off the pLys backbone. Transfection activity was highest 48 h after sample introduction, and PEG 3400 was determined to be the most favorable spacer size tested. pLys-PEG-folate:DNA transfection was also found to be both concentration dependent and saturable; plus, it was blocked by the addition of excess-free folate, indicative of a specific mechanism of uptake. Transfection activity was virtually identical for complexes formed in 10% serum-supplemented media, deionized water, or Hepes buffer. And, cell viability remained greater than 85% at the highest concentrations of pLys-PEG-folate:DNA complexes tested (4.8 µg/mL pLys 331 000; 12 µg/mL DNA). Taken together, these observations provide evidence that pLys-PEG-folate:DNA complexes are taken up specifically by the folate endocytosis pathway, and that the intramolecular spatial distance of the ligand from the pLys backbone dramatically influences transfection.

INTRODUCTION

Poly-L-lysine (pLys)1 is one of many polycations used to condense plasmid DNA through electrostatic interactions (1, 2). Electron microscopic examination of pLys: DNA complexes has revealed that nanometer-sized particles, either as solid or toroidal conformations, form as the result of electrostatic complexation (3). Because these diameters closely approximate the diameter of coated pits found on the plasma membrane of endocytosing cells, pLys condensation of plasmid DNA became a popular technique for use in DNA transfection studies (3, 4). Unfortunately, DNA (or oligonucleotide) condensation with pLys alone did not necessarily ensure successful transfection (4-6), probably because pLys complexes relied on nonspecific adsorptive pinocytosis mechanisms for cellular entry (7, 8). To overcome the problems associated with poor cellular uptake of pLys:DNA complexes, a portion of the -amino side chains on the pLys backbone has been derivatized with cell-specific targeting ligands. Thus, following condensation with DNA, externally oriented ligands on the pLys backbone became available for binding to specific cell surface receptors. Transferrin, insulin, asialoglycoprotein, monoclonal antibodies, mannose, lactose, and * To whom correspondence should be addressed. Endocyte Pharmaceuticals, Inc., 1205 Kent Ave., West Lafayette, IN 47906. Phone: (765) 463-7175. Fax: (765) 463-9271. E-mail: [email protected]. † GlaxoWellcome Research Institute. 1 Abbreviations: FFMEM, folate-free Dulbecco’s modified Eagles medium; Hepes, N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid; NHS-PEG-MAL, N-hydroxysuccinimidyl-poly(ethylene glycol)-maleiimide; MTT, 3-(4,5-dimethylthiazol-2-yl)2,5-diphenyltetrazolium; NHS, N-hydroxysuccinimide; PBS, phosphate-buffered saline; pLys, poly-L-lysine; PEG, poly(ethylene glycol).

folic acid have all been shown to improve cellular uptake of pLys:DNA (or pLys:oligonucleotide complexes) into cells (3, 4, 6, 8-19). However, the majority of the delivered DNA was believed to remain sequestered within endosomes/lysosomes, hence, unavailable for transfection (7, 8). Fortunately, improvements such as the incorporation of fusogenic peptides (20), perfringolysin O (21), or replication-incompetent adenovirus (13, 14, 17, 22, 23) into the overall pLys:DNA delivery vehicle have greatly enhanced transfection levels. Despite these improvements, the orientation of the ligand on the pLys backbone has never been optimized. Conceptually, the ligand should be (i) firmly anchored to the pLys backbone (preferably via a covalent bond), (ii) anchored in an orientation that does not destroy its affinity for its receptor, and (iii) positioned far enough away from the pLys backbone to allow good penetration into the binding pocket of its cell surface receptor. Interestingly, the latter concern may be of little importance when very large ligands (like insulin, transferrin, asialoglycoprotein, etc.) are employed for targeted pLys: DNA delivery. However, smaller ligands attached to pLys (folates, mannose, etc.) may not be effectively presented in an orientation that permits optimal receptor binding, especially when DNA is present and the overall complex is highly condensed. In this publication we describe a method for transfecting plasmid DNA into cultured cells using a pLys-based delivery vehicle that contains an extended ligand spacer arm. The technique involves the use of a poly(ethylene glycol) (PEG) “spacer” which is derivatized with a ligand to one end and pLys to the other end. Folate was chosen as the ligand of choice for this investigation owing to its relative ease of chemical derivatization and remarkable ability to deliver covalently attached macromolecules and/ or delivery systems into receptor-bearing cells (6, 17, 24-

10.1021/bc990066n CCC: $18.00 © 1999 American Chemical Society Published on Web 11/15/1999

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38). Using a plasmid containing a luciferase reporter gene, we were able to quantitatively evaluate transfections mediated by various pLys-PEG-folate:DNA complexes. Importantly, this technique was further optimized by carefully evaluating the effect of complexation charge ratios, complexation medias, PEG size, and the extent of pLys derivatization. EXPERIMENTAL PROCEDURES

Preparation of Folate-γ-Cysteine. Folate-γ-cysteine (pteroyl-γGlu-Cys) was synthesized as previously described.2 Briefly, an Applied Biosystems Inc. 433A peptide synthesizer equipped with conductivity monitoring was used to prepare folate-γ-cysteine at 0.25 mmol scale. The compound was formed by the stepwise addition of Fmoc-Glu-OtBu followed by N10-trifluoracetylpteroic acid onto a cysteine-modified p-hydroxymethylphenoxymethyl polystyrene resin. Following a wash with excess dichloromethane, the resin was repeatedly treated with 0.1 M aqueous piperidine (to remove the N10-trifluoracetyl group) and then with a cleavage/deprotection cocktail consisting of 750 mg of phenol, 10 mL of trifluoroacetic acid, 0.5 mL of water, 0.25 mL of dithioethane, and 0.5 mL of thioanisol for 2 h at room temperature. The freely soluble compound was separated from the resin by filtration through a medium pore fritted funnel. The filtrate was then diluted with 4 vol of diethyl ether to precipitate the folate-γ-cysteine product. Following multiple ether washes, the precipitate was dried in vacuo. Barring no further purification, folate-γ-cysteine produced in this manner was typically 70-85% pure, as determined by the A280 peak area generated from C18 reversed-phase HPLC analyses, with the primary impurity being the dimerized (disulfide-linked) species. An Ellman’s assay (39) on the crude material confirmed that the solid contained 70-85% free sulfhydryls. Preparation of NHS-PEG-γ-Folate. A total of 5.9 µmol of commercially available or custom-synthesized N-hydroxysuccinimidyl-poly(ethylene glycol)-maleiimide (NHS-PEG-Mal; all of these PEG reagents along with their analytical characterization data were provided by Shearwater Polymers, Huntsville, AL) were dissolved in 1 mL of dry dichloromethane within a 10 mL roundbottom flask. A 3-fold molar excess of solid folate-γcysteine was added to the flask. While stirring, a 5-fold molar excess (relative to folate-γ-cysteine) of neat triethylamine was added to the flask, and the solution was stirred continuously for a minimum of 18 h at room temperature. Under these conditions, thiols can add to maleiimide groups in organic solvents (40).3 Unreacted folate-γ-cysteine remained a solid while NHS-PEG-γfolate had completely dissolved. Samples were centrifuged for 5 min at 15000g, and the yellow supernatants were diluted with 50 mL of diethyl ether to precipitate the NHS-PEG-γ-folate product. Samples were spun at 2500 rpm for 10 min, the supernatants were decanted, and the yellow pellets were redissolved in 1 mL of dichloromethane. The ether precipitation step was repeated two more times, and the final yellow pellet was dissolved in 1 mL of chloroform and stored at -20 °C. The concentration of NHS-PEG-γ-folate was determined as follows. A 10 µL aliquot of the chloroform sample was placed in a 1 mL eppendorf tube and dried using a Savant 2

J. Drug Targeting (1999) (in press). We have also successfully prepared this reagent in dimethyl sulfoxide; however, an additional ether precipitation step and selective solvation in DCM is needed prior to the final workup. 3

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tabletop speedvac. The dried residue was dissolved in 1 mL of phosphate-buffered saline (PBS), and the absorbance at 362 nm was measured. Molar concentrations were determined using the extinction coefficient for folate at 362 nm (362 ) 6100 M-1 cm-1). Preparation of pLys-PEG-γ-Folate. Stock solutions of poly-L-lysine hydrobromide (Sigma Chemical, St. Louis, MO) with an average molecular weight of 331 000 were prepared in 100 mM N-2-hydroxyethylpiperazine-N′-3propanesulfonic acid, pH 8.0. Typically, 3 nmol of pLys were added to a 1.5 mL eppendorf tube, and additional buffer was added to give a final volume of 450 µL. An aliquot of NHS-PEG-γ-folate (in CHCl3) was added to a 1.5 mL eppendorf tube. The CHCl3 was removed using a speedvac, and the remaining residue was hydrated in 50 µL of deionized water. The entire volume of NHS-PEGγ-folate solution was then mixed with the pLys sample (while vortexing), and the sample was left to react at room temperature for a minimum of 2 h. Unreacted NHSPEG-γ-folate was removed from pLys-PEG-γ-folate by exhaustive dialysis through a 30 000 molecular weight cutoff membrane using 20 mM N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid (Hepes) and 100 mM NaCl, pH 7.30. Alternatively, conjugates were purified by using a Mono S 5/5 column (buffer ) 50 mM Hepes, pH 7.9, and a NaCl gradient from 0 to 3 M over 30 min) followed by overnight dialysis against 20 mM Hepes and 100 mM NaCl, pH 7.3. Samples were filtered with a 0.22 µm membrane prior to characterization (vide infra). Characterization of pLys-PEG-γ-Folate Conjugates. Poly-L-lysine content was measured using a modified trypan blue precipitation assay (41). Aliquots from a stock 1.0 mg/mL solution of pLys (mw 331 000) in deionized water were dispensed into 1.5 mL eppendorf tubes. PBS was added to each tube to bring the final volume to 990 µL. Similarly, sample aliquots estimated to contain approximately 5 µg of pLys were added to eppendorf tubes and diluted with PBS to 990 µL. A total of 10 µL of a 4 mg/mL trypan blue solution was added to each tube followed by vortexing for 5 s each. Tubes were heated to 60 °C and shaken for a minimum of 1 h. Following immersion in ice for 5 min, tubes were then spun for 5 min at 15000g. The absorbance at 580 nm of each supernatant was measured, and the concentrations of pLys were calculated from a standard curve.4 Since each PEG moiety present in the pLys-PEG-folate conjugate contains one folate moiety, the PEG content from each sample was determined by measuring the absorbance due to folate at 362 nm.5 An estimate of the degree of PEG substitution on the pLys molecule was calculated by simply dividing the concentration of PEG by the concentration of pLys. Cell Culture. HeLa and IGROV-1 cells were grown continuously as a monolayer using folate-free Dulbecco’s modified Eagles medium (FFMEM) containing 10% heatinactivated fetal calf serum and 2 mM L-glutamine at 37 °C in a 5% CO2/95% air humidified atmosphere. Because the heat-inactivated serum contained its normal complement of endogenous folates, the cells could be maintained indefinitely in the folate-deficient medium (24). Supercoiled Plasmid DNAs. Plasmids pGL3 (for luciferase gene expression; Promega, Madison, WI) and pSV-βgal (for β-galactosidase expression; Promega) were transformed and propagated in Escherichia coli strain 4 Neither folic acid nor PEG 3400 caused Trypan Blue to precipitate under similar conditions. 5 At the concentrations measured, pLys does not appreciably absorb at 362 nm.

Folate Copolymer Transfection

JM109. Milligram quantities of super-coiled plasmids were prepared using Quiagen plasmid preparation kits, as suggested by the manufacturer. Formation of pLys-PEG-folate:DNA Complexes. Complexes were formed in either polypropylene tubes or polystyrene plates. Briefly, pLys was dissolved in half the final volume of either reagent-grade water, Hepes, or 10% serum-supplemented FFMEM. Plasmid DNA was then dissolved in the second half of water, Hepes, or serum-supplemented FFMEM, and the entire solution was mixed with the pLys solution. Plasmid DNA concentrations were typically 20 µg/mL at this step. Following a 5 s vortex mix, the solution was left to complex at room temperature for 1 h. The solution was later diluted with additional serum-containing FFMEM and added to cells without further purification. Time-Dependent Transfection. Cells were added to 96-well plates in a total volume of 100 µL of 10% serumsupplemented FFMEM, and the plates were incubated at 37 °C for 18-24 h. Spent incubation media was replaced with 100 µL of sample-containing media per well (four wells per sample), and cells were incubated at 37 °C for the indicated times. Each well was vacuum aspirated, and 20 µL of 1× Cell Lysis Buffer (Promega) was added per well. Plates were then stored overnight at -70 °C. Following a 30 min thaw at room temperature on a rocker platform, cell lysates were quantitatively transferred to wells of Dynatech Microlite plates. A Dynatech ML3000 luminometer was used to autodispense 100 µL of luciferase assay reagent (Promega)/well and to sum the relative luminescence units over a 30 s period. Ratio Study. Cells were added to 96-well plates in a total volume of 100 µL of the media described above, and the plates were incubated at 37 °C for 18-24 h. Spent incubation media was replaced with 100 µL of samplecontaining media/well (three wells per sample), and cells were incubated at 37 °C for 48 h. Importantly, the quantity of plasmid DNA was held constant at 1 µg/mL in each well, whereas the quantity of pLys or pLysPEG3400-folate was varied from 0 to 4 µg/mL to account for the various (ratios examined. Following the incubation, cells were harvested and the luminescence activity was measured according to the procedure detailed above. Concentration-Dependent Transfection and Related cytotoxicity. A total of 2000 HeLa cells in 100 µL of serum-supplemented FFMEM was added to each well of duplicate 96-well plates and then incubated at 37 °C for 18-24 h. Aliquots from a master pLys-PEGfolate:DNA complex mix (20 µg/mL as DNA) were added to individual polypropylene tubes and diluted with serum-supplemented FFMEM to the indicated concentrations. Spent incubation media in each well was replaced with 100 µL of the appropriately diluted complex (four wells per sample), and the cells were incubated for 48 h at 37 °C. Cells from one plate were harvested and the luminescence activity was measured according to the procedure detailed above. Cells in the duplicate plate were used in a cytotoxicity assay (two wells per sample). Briefly, 10 µL of a 5 mg/ mL 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) solution in PBS was added to each well 2 h prior to the end of the 37 °C incubation. At the end of the incubation, spent media was aspirated from each well, and 100 µL of dimethyl sulfoxide plus 10 µL of 0.1 M glycine, pH 9.0, was added per well. The absorbance at 580 nm was read for each well, and the percent

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cytotoxicity was calculated based on the absorbance values measured in wells that had contained untreated cells. Cytochemical Staining for β-Galactosidase. Approximately 50 000 cells in 2.5 mL of serum-supplemented media were added to each well of a Falcon 6-well plate and then incubated for 24 h at 37 °C. Using pSVβgal as the DNA source, pLys-PEG-folate:DNA complexes were prepared 1 h prior to the experiment according to the protocol described above. Spent incubation media was removed from each well and replaced with 1 mL of sample-containing, serum-supplemented media. Following a 48 h incubation at 37 °C, monolayers were rinsed twice with PBS and then stained for β-galactosidase activity using the protocol and reagents supplied in the β-galactosidase Staining Kit (In Vitrogen; San Diego, CA). Cells were stained for a maximum of 24 h and then photographed at 200× magnification using Kodak color slide film. Comparison of pLys-PEG-Folate:DNA Transfection to Cationic Liposome Transfection. A total of 2000 HeLa cells in 100 µL of serum-supplemented FFMEM was added to each well of a 96-well plate and then incubated at 37 °C for 18-24 h. pLys-PEG3400-folate: pGL3 complexes were formed per the method detailed above using 10% serum-supplemented FFMEM. The cationic liposomes were complexed with pGL3 in 10% serum-supplemented FFMEM using the average of the vendor-recommended quantity of liposome per unit DNA. Thus, 10 µg of Lipofectin (Life Technologies, Gaithersburg, MD), 12.5 µg of LipofectAmine (Life Technologies), 6 µg of DOTAP (Boehringer Mannheim, Indianapolis, IN), 3 µg of DOPSER (Boehringer Mannheim), 5 µg of Transfectam (Promega), or 4.7 µg of TFx-50 (Promega) were complexed/µg of pGL3. Spent incubation media in each well was replaced with 100 µL of the appropriately diluted complex (8 wells/sample; 100 ng of DNA/well), and the cells were incubated for 48 h at 37 °C. Following the incubation, cells were harvested and the luminescence activity in two-thirds of the cell lysate was measured according to the procedure detailed above. The remaining cell lysate was used to perform a DotMetric protein assay (GenoTechnologies, St. Louis, MO), per the manufacturers instructions. Luminescent activity was normalized by the protein content for each sample, and the results were expressed as relative luminescence units per milligram of cell protein. RESULTS

Proposed Structure of pLys-PEG-Folate:DNA Complexes. Figure 1 depicts a proposed structure for pLys-PEG-folate:DNA complexes. Here we assume that polyanionic DNA will electrostatically complex with polycationic pLys to form a charge-neutral hydrophobic core, and despite a covalent linkage to pLys, we further believe that the hydrophilic PEG-folate moieties should naturally orient outward from the core and into the aqueous milieu, thus forming a targetable micelle-like DNA delivery vehicle. Interestingly, a similar micellar structure has previously been proposed for oligonucleotide-polyoxyethylene derivatives (42); however, that particular delivery system lacked a cell-targeting ligand component. Time-Dependent Transfection. The first transfection parameters we examined were the effect of folatecopolymer transfection with respect to the plated cell density and the length of incubation (exposure) time. Cells were plated at increasing densities into the wells

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Figure 1. Proposed structure of pLys-PEG-folate:DNA complexes. Polyanionic DNA is assumed to electrostatically complex with polycationic pLys to form an electrically neutral, or hydrophobic core. The hydrophilic PEG-folate moieties naturally orient outward from this core into the bulk aqueous milieu, thus creating a targetable micelle-like DNA delivery vehicle.

of tissue-culture-treated microtitier plates and incubated for 18-24 h. Complexes between pLys-PEG3400-folate (72 PEG-folates/pLys chain) and plasmid DNA (1:1 ( charge ratio) were preformed in 10% serum-supplemented

Leamon et al.

FFMEM, and 100 ng of the complex (relative to DNA) was added to the wells. Following a 37 °C incubation for various time intervals, cells were harvested and assayed for luciferase activity. As shown in Figure 2, luciferase activity was not detected until >12 h of incubation, and this phenomenon was independent of cell density. However, the time of maximal luciferase expression did appear to be dependent on cell density. For example, at cell densities of >2000/well maximal luciferase activity was detected after 48 h of incubation. In contrast, maximal luciferase activity was reached and transiently sustained after 72 h for cells plated at the lower cell densities, suggesting that the extent of transfection (luciferase activity levels) was directly related to the degree of cell confluency within the wells. Interestingly, no luciferase activity was measured from cells treated with pLys:DNA (Figure 2) or pLys-PEG control complexes (not shown). Thus, for the remainder of the experiments described below, we chose to test cells plated at an initial density of 2000 cells/100 µL/well and to assess the levels of transfection 48 h later. Charge Ratio Optimization. Theoretically, an electrostatic complex between a polycation and polyanion would carry a net neutral charge if the ( charge ratio were unity. A neutral complex would be desirable to incorporate into a ligand-targeted nucleic acid delivery vehicle, such as the one proposed here (see Figure 1), because it would eliminate the opportunity for nonspecific, adsorptive binding and uptake to nontarget cells. Unfortunately, interactions with buffer-associated ions and/or component proteins (e.g., from the serum) may alter the thermodynamically optimal ( charge ratio that would promote the formation of a neutral particle. Therefore, we elected to experimentally determine the optimal ( complexation ratios for pLys-PEG-folate and plasmid DNA, while simultaneously exploring the effect of the complexation media. Complexes were prepared between pGL3 (5256 base pair) and increasing charge equivalents of pLys or pLysPEG3400-folate (72 PEG-folates per pLys chain), thereby spanning a large ( charge ratio spectrum.6 Cells were exposed to the complexes for 48 h, and luciferase activity

Figure 2. Effect of cell density and exposure time on pLys-PEG-folate:DNA transfection. A total of 100 µL of 10% serum-supplemented FFMEM containing pLys-PEG3400-folate:DNA complexes (72 PEG-folates per pLys chain) was added to HeLa monolayers of increasing cell densities in 96 well plates. Cells were incubated at 37 °C for the indicated times. Spent incubation media was removed, and cells were assayed for endogenous luciferase activity as described in the Experimental Procedures. Number of cells originally seeded per well of a 96 well plate: (b, O) 500 cells; (9, 0) 1000 cells; (2, 4) 2000 cells; (1, 3) 4000 cells. Solid symbols represent pLys-PEGfolate:DNA complexes, while open symbols represent pLys:DNA complexes. Error bars represent one standard deviation (n ) 4).

Folate Copolymer Transfection

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Figure 3. Effect of ( charge ratio and complexation medium on pLys-PEG-folate:DNA transfection. Complexes were prepared between pGL3 and increasing charge equivalents of pLys-PEG3400-folate (72 PEG-folates per pLys chain). The quantity of plasmid DNA was held constant at 1 µg/mL in each well, whereas the quantity of pLys varied from 0 to 4 µg/mL to account for the various ( ratios examined. Cells were exposed to these complexes for 48 h, and endogenous luciferase activity was measured according to Experimental Procedures. (Solid bars) pLys-PEG-folate:DNA complexes; (open bars) pLys:DNA complexes. (A) Complexes formed in FFMEM supplemented with 10% heat-inactivated fetal calf serum; (B) Complexes formed in double deionized water. Error bars represent one standard deviation (n ) 3).

was then measured. The results in Figure 3A show that luciferase activity levels gradually increased with respect to an increasing ( charge ratio until a maximum was reached at a near-neutral ratio. Beyond this optimum, the luciferase activity decreased as the charge ratio was further raised. In contrast, pGL3 titrated with unmodified pLys produced relatively large particles (easily observed at 250× magnification floating in the media) that were very ineffective transfection agents. This may partly explain why only minimal transfection activity was measured with these particles and only at the higher ( ratios (Figure 3). All of the data presented thus far have been generated using complexes formed in 10% serum-supplemented media. This was initially done to optimize our experimental conditions for future cell studies that were 6 Approximately four pLys chains or four pLys-PEG 3400-folate copolymer units are needed to charge-neutralize 1 plasmid molecule.

intended to be performed in the presence of serum. However, we decided to determine if complexes preformed in nonserum-containing solutions were more efficient mediators of transfection. Thus, we chose to titrate pGL3 with increasing charge equivalents of pLysPEG-folate in deionized water. As shown in Figure 3B, maximal levels of luciferase activity were achieved with a charge ratio near unity, similar to the results obtained from complexes formed in the presence of 10% serum. Interestingly, identical results were obtained when complexes preformed in Hepes-buffered saline solution were used in a similar assay (data not shown). Taken together, we interpret these results to suggest that optimal pLysPEG-folate:DNA complexes are formed when the ( charge ratio is near unity and that the complexation process is not significantly influenced by the composition of the complexation buffer. Effect of Increasing PEG Size. Figure 1 depicts a cartoon of a possible organizational structure formed between pLys-PEG-folate and nucleic acids. The PEG

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Figure 4. Effect of PEG size on pLys-PEG-folate:DNA transfection. Cells were incubated for 48 h at 37 °C in the presence of various pLys-PEG-folate:pGL3 complexes (1 µg/mL as DNA). Following the incubation, cells were harvested and the endogenous luminescence activity was measured according to Experimental Procedures. Control represents a pLys:DNA complex (no folate). Values 0-20000 represent the average molecular weight of PEG used in the particular pLys-PEG-folate conjugate assayed. (A) HeLa cells. (B) IGROV-1 cells. Error bars represent one standard deviation (n ) 4).

component is assumed to orient on the outermost shell of the complex and to interact with the external aqueous environment, similar to the PEG component of Stealth liposomes (43, 44). However, it is not known if transfection via pLys-PEG-folate:DNA complexes is dependent on the length of the PEG chain utilized. To evaluate this effect, we derivatized pLys with folate attached to PEG of various sizes ranging 0-20000 in average molecular weight. Complexes formed between this collection of pLys-PEG-folates and pGL3 were incubated with cultured cells for 48 h, and the levels of luciferase activity were measured. As shown in Figure 4A, very low levels of luciferase activity were measured in HeLa cells treated with complexes containing PEG components of 2000 molecular weight and smaller. However, significant luciferase activity was measured from cells transfected with PEG3400-containing complexes. There was also no apparent advantage using the higher molecular weight PEG components (i.e., PEG10000 or PEG20000). Interestingly, a similar PEG molecular weight trend was observed when a second cell type (IGROV-1) was used in this assay (see

Figure 4B). This phenomenon is likely related to the observation that PEG forms “mushroom-like”, or compacted, conformations at higher molecular weights (44), indicating that steric protection of the pLys:DNA core is a desirable property for transfection (see Figure 1). Notably, the remainder of the experiments described below was completed using pLys-PEG3400-folate. Effect of PEG-Folate Substitution. In addition to determining a favored PEG size for pLys-PEG-folate: pGL3 transfection, we investigated the effect of increasing the number of PEG-folate units conjugated per pLys chain. Thus, pLys was conjugated with increasing amounts of NHS-PEG3400-folate, and following purification, the extent of PEG3400-folate derivatization was determined. Complexes were formed with pGL3 and then added to HeLa cells for 48 h. As shown in Figure 5, a conjugate constructed with an average of 72 PEG3400-folates per pLys chain (2.8% of the ∼2586 lysine units derivatized) produced the highest measured level of luciferase activity in HeLa cells. Although no further attempt was made to determine the exact derivatization optimum, we believe

Folate Copolymer Transfection

Figure 5. Effect of PEG substitution on pLys-PEG3400-folate: DNA transfection. pLys (MW ≈ 331 000) was derivatized with increasing amounts of PEG3400-folate and complexed to pGL3. Cells were incubated for 48 h at 37 °C in the presence of 1 µg/ mL complex (as DNA). Following the incubation, cells were harvested and the endogenous luminescence activity was measured according to Experimental Procedures. Error bars represent 1 standard deviation (n ) 4).

Figure 6. Concentration-dependent transfection. pLys-PEG3400folate:pGL3 complexes (72 PEG-folates/pLys chain) were formed and diluted in serum-supplemented FFMEM and then added at increasing concentrations to HeLa cell monolayers. Following a 48 h 37 °C incubation, cells were harvested and the endogenous luminescence activity was measured according to Experimental Procedures. (b) pLys-PEG3400-folate:pGL3 complex; (O) pLys:pGL3 complex. Error bars represent 1 standard deviation (n ) 4).

one clearly exists. Furthermore, the optimum PEG derivatization number may likely dictate the overall threedimensional structure (and transfection potency) of these pLys-PEG-folate:DNA complexes. Concentration-Dependent Transfection and Related Cytotoxicity. The folate moiety within the pLysPEG-folate:DNA complex should effectively bind to external folate binding protein receptors on HeLa cells as well as mediate uptake via endocytosis. Because receptormediated endocytosis is a saturable process, we elected to determine the concentration dependence of pLys-PEGfolate:DNA transfection. As shown in Figure 6, luciferase activity reached a plateau at pLys-PEG3400-folate:DNA concentrations of 8 µg/mL and higher (relative to DNA). Similar to what we observed in Figure 3 above, complexes formed with pLys had absolutely no transfection capability, even with concentrations as high as 12 µg/mL. Plus,

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Figure 7. Cytotoxicity of pLys-PE2G3400-folate:pGL3 complexes. Cells in a duplicate 96 well plate to that described in the legend to Figure 6 were used to evaluate the cytotoxicity of pLys-PEG3400-folate:pGL3 complexes. Thus, complexes were formed and diluted in serum-supplemented FFMEM and then added at increasing concentrations to HeLa cell monolayers. Following a 46 h 37 °C incubation, cells were incubated for an additional 2 h at 37 °C in the presence of the MTT viability stain. Cells were harvested, and the percent cytotoxicity was calculated based on the viability values measured in wells that had contained untreated cells, as detailed in the Experimental Procedures. (Solid bars) pLys-PEG3400-folate:pGL3 complexes; (open bars) pLys:pGL3 complexes. Error bars represent 1 standard deviation (n ) 2).

a 100-fold excess of free folic acid nearly abolished (activity reduced by 95% at 5 µg/mL complex; not shown) pLys-PEG3400-folate:DNA transfection, thus confirming the involvement of a receptor-mediated process. Because there are reports that pLys:DNA complexes are toxic to some cells (45, 46), we chose to examine the extent of cell toxicity on cells treated with increasing concentrations of pLys-PEG3400-folate:pGL3 complexes. As shown in Figure 7, no significant cytotoxicity was measured in our HeLa cells treated with complexes up to 4.8 µg/mL pLys 331 000 molecular weight (or 12 µg/ mL DNA). Cytochemical Staining for β-Galactosidase. To better assess the extent to which treated cells are transfected by pLys-PEG-folate:DNA complexes, we elected to quantify the number of transfected cells in a population by using a β-galactosidase reporter plasmid and chromogenic staining. Complexes between pSV-βgal and pLys-PEG3400-folate (72 PEG-folates/pLys chain) were formed in 10% serum-containing FFMEM per the usual protocol detailed above. A saturable concentration of the complex (9 µg, relative to DNA) was introduced to adherent cells in a 6-well Falcon culture dish, and the plate was incubated at 37 °C for 48 h. Using a commercially available kit, functionally transfected (i.e., β-galactosidase expressing) cells were detected by the visual appearance of a blue stain. Figure 8 shows a representative field of cells treated in this manner. Interestingly, positive blue staining varied among the cells. Some cells stained very heavily, and the stain was diffuse throughout the entire cell body. Other cells, however, limited their display of blue staining to vacuolar compartments. By summing the total number of cells containing blue stain (both vacuolar and diffuse) within any given field of view, we estimated that approximately 6% of the exposed cells were functionally transfected by pLys-PEG3400-folate:pSV-βgal complexes (see Discussion). Comparison of pLys-PEG-Folate:DNA Transfection to Cationic Liposome Transfection. Cationic liposomes have been widely used to transfect plasmid

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Leamon et al. DISCUSSION

Figure 8. Cytochemical staining for β-galactosidase activity. Complexes of pSV-βgal and pLys-PEG3400-folate (72 PEG-folates/ pLys chain) were formed in 10% serum-containing FFMEM for 1 h, as described in Experimental Procedures and were then added to plated cells at saturable levels (9 µg with respect to DNA). Following 48 h of incubation at 37 °C, cells were developed using a commercially available β-galactosidase staining kit. Functionally transfected cells (blue color) were identified using bright-field microscopy. The transfection efficiency was calculated by counting the number of stained cells in a random microscope field viewed at 200× and then dividing by the total number of cells in that same field. Four fields were counted and averaged to give an overall transfection efficiency of ∼6%.

DNA into cells both in vitro and in vivo (47-50). Unfortunately, cationic liposome-mediated transfection has not worked well in the presence of serum (50, 51). Because our pLys-PEG-folate conjugates are typically complexed to plasmid DNA in the presence of serum, we elected to evaluate the levels of cellular transfection achieved by pLys-PEG-folate:DNA complexes to that obtained with cationic liposome:DNA complexes also prepared in the presence of 10% serum. The results in Figure 9 show that pLys-PEG3400-folate (72 PEG-folates/ pLys chain) produced equal to or greater levels of transfection relative to that of six popular commercially available cationic liposome formulations, each tested using their vendor-recommended optimal complexation ratios (∼2.5-5 ( ratios; see Experimental Procedures). Importantly, the data in this figure were normalized with respect to the amount of residual cellular protein present at the end of the experiment. Many of the cationic liposome formulations produced significant cell killing (which was evident from the lower total cell protein values), whereas pLys-PEG3400-folate complexes did not affect cell number (see Figure 7). Considering the added benefit of cell targeting via folate endocytosis, pLysPEG3400-folate was obviously the preferred transfection reagent for use in these studies that employed serumcontaining medias.

We developed a method for transfecting plasmid DNA into cultured cells using a targetable pLys-based delivery vehicle that contains an extended poly(ethylene glycol) “spacer” arm strategically placed between the ligand and the pLys backbone. Using a luciferase reporter gene plasmid, we quantitatively evaluated transfections mediated by various pLys-PEG-folate:DNA complexes and carefully optimized this technique by studying the influence of complexation charge ratios, complexation medias, PEG size, and the extent of pLys derivatization. We discovered that a finite lag period exists between the time that pLys-PEG-folate:DNA complexes enter the cells and the time that endogenous luciferase activity is detected (Figure 2). This latency period is crudely estimated to fall within 12-24 h postexposure to cells. Interestingly, folate-toxin conjugates have been shown to gain access to the cytosol after only a 2-8 h delay following entry into cells via receptor-mediated endocytosis (25, 27). Perhaps the longer lag phase seen with our pLys-PEG-folate:DNA complexes is due to (i) the larger size of the “payload” (i.e., condensed plasmid) and (ii) the fact that this payload must further enter the nucleus, become transcribed and then translated prior to the detection of transfection activity. Another interesting observation is that the time of maximal luciferase expression appeared to be dependent on cell density (see Figure 2). Cells in the most heavily seeded wells displayed maximal luciferase activity at the earlier time points, while cells in the lower seeded wells reached maximal luciferase activity at the later time points. Interestingly, this phenomenon may be related to a recent observation that wild-type folate receptors failed to deliver externally bound folate molecules into the cytoplasm of confluent, nondividing cells (52). Assuming this physiological effect occurred in our HeLa cells, one might have predicted that the more heavily seeded wells (4000 cells/well; Figure 2) would have reached confluency, or folate-endocytosis incompetentcy, faster than the lesser-seeded wells; this, in turn, would have negatively affected the luciferase activity levels (especially at the later time points). Overall, this celldensity effect may have ramifications for comparing similar results of different studies and, therefore, should be cautiously noted. During the course of this study, we discovered that the size of PEG used to construct the pLys-PEG-folate conjugate was critical for successful mediation of DNA transfection into target cells. Thus, the results in Figure 4 show that no enhancement of DNA transfection was detected until the PEG size within the pLys-PEG-folate conjugate was increased to 3400 in molecular weight. PEGs larger than 3400 molecular weight added little (if any) benefit to the overall transfection activity, and that effect varied among the two cell types tested within this study. This linker or “cushion” effect has also been seen with Stealth liposomes, where lipids modified with PEG of molecular weights >2000 had diminished protective properties (44). We believe, however, that the PEG within pLys-PEG-folate conjugates serves not only to provide a hydrophilic barrier to protect the pLys-condensed DNA but that it also acts as an extended linker arm to allow the folate ligand to optimally bind to its cell-surface receptor (see Figure 1). Notably, extended linker arms have also been shown to be critically beneficial for the successful targeting of folate liposomes (30). In particular, folate anchored to the phospholipid headgroup by a oneor two-amino acid “spacer” failed to promote targeted

Folate Copolymer Transfection

Bioconjugate Chem., Vol. 10, No. 6, 1999 955

Figure 9. Comparison of pLys-PEG3400-folate and cationic liposome mediated DNA transfection. pGL3 was complexed to pLysPEG3400-folate (72 PEG-folates/pLys chain) or to preformed cationic liposomes in 10% serum-supplemented FFMEM for 10-30 min at room temperature. Complexes were added to cells (1 µg/mL as DNA) and incubated for 48 h at 37 °C. Spent incubation media was removed, and cells were assayed for endogenous luciferase activity as described in Experimental Procedures. Error bars represent 1 standard deviation (n ) 8).

liposome delivery to cells, whereas a ∼3400 molecular weight PEG spacer produced fully targetable liposomes (30). Interestingly, this present study revealed that a useful PEG size for the pLys-PEG-folate transfection is also 3400 in molecular weight. And although we do not yet fully understand why the PEG 2000 spacer (or shorter) failed to mediate folate-targetable transfection, it is possible that steric constraints between the folate receptor and our pLys-PEG-folate:DNA particles prevented effective ligand binding. However, this parameter should be investigated further. The value of including the PEG spacer within a targeted pLys:DNA complex was further revealed when we tested the ability of pLys-folate to transfect cells in parallel with pLys-PEG3400-folate. The data presented in Figure 4 shows that pLys-folate is an inferior transfection agent. The simple addition of a PEG spacer afforded a 74-fold enhancement of transfection in HeLa cells and a 10-fold enhancement in IGROV-1 cells, relative to transfection levels mediated by pLys-folate. Presumably, the distinct levels of transfection enhancement between these two cell lines is due to their distinct levels of folate receptor expression (>15 × 106 folate binding sites/HeLa cell versus ∼5 × 105 sites/IGROV-1 cell) (26, 53). Interestingly, others have reported that transfection via pLysfolate:DNA complexes could be enhanced by co-treatment of cells with lysosomotropic agents (8) or adenoviral particles (17). Although not tested here, these techniques may also improve upon the transfection mediated by pLys-PEG-folate. The proposed structure of pLys-PEG-folate:DNA complexes shown in Figure 1 represents what we conceive to be an effective transfection particle. It seemed logical to assume that polycationic and polyanionic polymers can, under charge neutral conditions, complex together to form a hydrophobic “core”. Indeed, such a hypothesis has recently been proposed for oligonucleotide-polyoxyethylene complexes (42). And in a related way, it seemed logical to assume that the hydrophilic PEG-folate moiety

would orient itself outward toward the bulk solution. This is evidenced by the observations that (i) free folic acid blocks the cell-association of the pLys-PEG3400-folate:DNA complex and (ii) the transfection process mediated by pLys-PEG3400-folate is saturable, suggesting that transfection occurs by a receptor-mediated process. Obviously, further biochemical analyses as well as electron microscopic characterization of these complexes is needed to confirm the proposed structure and to determine the effect of varying the size of the pLys component within the complex (54). All of the studies presented above were conducted on cultured cells previously determined to express folate binding protein receptors. Assuming that each cell in the culture bears this receptor, one might expect that each cell would become transfected by pLys-PEG3400-folate: DNA complexes. However, when we attempted to stain for transfection following the delivery of a plasmid containing a β-galactosidase gene, only 6% of these treated cells stained positively (see Figure 9). It is very unlikely that 94% of the cell population was incompetent to bind and internalize these complexes, since delivery of folate-toxin conjugates into the identical cell line effectively killed the entire population (25, 27). Instead, we believe that the majority of the cells either expressed very low levels (staining not visually detectable) of β-galactosidase, or that the majority of DNA plasmid within these cells remained entrapped within organellar structures. Unlike protein toxins, plasmid DNA must breach not only the endosomal barrier but also the nuclear membrane barrier prior to eliciting an effect. Although access to the cytosol is difficult in itself, that alone would not guarantee successful translocation of DNA into the nucleus (55). Clearly, these results indicate that transfection mediated by pLys-PEG3400-folate may be further enhanced, possibly with the co-incorporation of endosome-disrupting agents (14, 20, 21).

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